1. Field of the Invention
The present invention relates to a microRNA-based short hairpin RNA, and a medicament comprising the said microRNA-based short hairpin RNA, particularly to a microRNA-based short hairpin RNA for silencing NR1 subunit of N-methyl-D-aspartate receptor, and an analgesic drug comprising the said microRNA-based short hairpin RNA.
2. Description of the Related Art
RNA interference refers to a phenomenon of post-transcriptional gene silencing being mediated by small double-stranded RNAs (also known as small interfering RNAs or siRNAs) that comprises endogenously encoded RNA to guide ribonucleoprotein complex, such as RNA induced silencing complex (RISA), to target message RNA (mRNA). The RNA interference is first discovered in 1990s in plants, the silencing mechanism thereof refers to exotic double-strand RNAs in cells being processed by Dicer and generating a plurality of siRNAs, with those siRNAs being self-unwound, targeting to particular messenger RNAs, further conducting RNA-induced silencing complex (RISC) to degrade the particular messenger RNAs, and finally resulting in post-transcriptional gene silencing on the said particular messenger RNAs.
In conventional arts, a siRNA, comprising 21 ribonucleic acids, has been developed, as reference to US publishing No. 2011/0263676 entitled “A SMALL INTERFERING RNA FOR GENE KNOCKDOWN OF THE SUBCUTANEOUS N-METHYL-D-ASPARTATE RECEPTOR NR1 SUBUNIT, AND IT'S APPLICATION ON PHARMACEUTICS,” to achieve analgesic effects without leading to any side effects, such as nausea, lethargy, faint and motor un-coordination, that cause by conventional NMDA receptor antagonists (e.g. ketamine). By delivering the said siRNA to affected parts through subcutaneously injection, a temporary RNA interfering on NR1 subunit of N-methyl-D-aspartate receptor in hypoderm is conducted, so as to achieve antinociceptive effects on the affected parts.
However, due to the instability of the said siRNA, only a small amount of the said siRNA can truly affect on the affected parts, and therefore, a high dosage and a pharmaceutical acceptable carrier is needed to effectively deliver the said siRNA into nucleus for achieving NR1 silencing and analgesic. Also, the silencing effect of the said siRNA only last for 7 days, with the analgesic efficiency thereof decreasing by days after 7 days. Accordingly, a frequently injection is necessary if a long-term analgesic treatment is requested.
For improving the above-identified disadvantages of the said siRNA, a conventional analgesic is developed, as reference to U.S. application Ser. No. 13/027,742 entitled “A SHORT HAIRPIN RNA FOR GENE KNOCKDOWN OF NR1 SUBUNIT OF THE N-METHYL-D-ASPARTATE RECEPTOR AND ITS APPLICATION ON PHARMACEUTICS,” and comprises a short hairpin RNA encoded in a pSilencer vector, being stable and easy to manufacture. The said short hairpin RNA has 45-65 nucleotides comprising a first fragment, a second fragment and a connection fragment, with the first fragment having homologous nucleotides among NR1 subunit of NMDA receptor, with the second fragment having complementary sequence to the first fragment, and with connecting fragment being 3-23 randomly arranged nucleotides. After delivering the conventional analgesic to affected parts, the said short hairpin RNA are transcribed by RNA polymerase III in cells, self-folded into a hairpin structure, and processed by endogenous nucleases to generate short double stranded RNAs, conducting a destruction of NR1 subunit of NMDA receptor medicated by RISC to achieve antinociception.
Although the said shRNA show lasting silencing effect (with duration of more than 14 days) on NR1 subunit of NMDA receptor in comparison with the said siRNA, it is poor in achieving immediately analgesic effect due to the late response of the said shRNA, with the antinociceptive effect thereof only presenting at least 7 days after cell-delivering. Therefore, the shRNA is failed to relieve acute pain. Furthermore, a dramatic dosage of a pharmaceutical acceptable carrier is needed for the said shRNA to reach a high delivering efficiency, and thus that the cost of the conventional analgesic in commercialization is high.
In 2005, Stegmeier et. al., have published a shRNA expression system (A Lentiviral microRNA-based System for Single-Copy Polymerase II-regulated RNA interference in mammalian cells. PNAS 102:13212-13217), to facilitate the tracking of shRNA production in cells, by providing a vector pPRIME to transcribe shRNAs under controls of RNA polymerase II and RNA polymerase III. The vector pPRIME provides sufficient penetrance for the use of encoded shRNAs. However, Stegmeier et. al. provides less information on practical uses of the said vector pPRIME and encoded shRNAs, especially in pharmaceutics.
Hence, it is necessary to improve the above-identified disadvantages and issues, and to provide an alternative strategy for achieving fast and lasting gene silencing on NR1 subunit of NMDA receptor, finally to accomplish fast and long-lasting antinociceptive effects and to put to use in pharmaceutics industry.